Project Summary Human implantation, like that of other species with hemochorial placentation, is highly invasive and results in an intermingling of maternal and fetal cells that persists for the duration of pregnancy. This conjoining of the two compartments is essential for establishing optimal communication, laying the foundation for healthy pregnancies and long-term health of both mothers and their children. The maternal-fetal interface traditionally has been defined as the sites of cellular contact between placental trophoblast and maternal endometrium and blood, expanding with advancing gestation. A newer view is that this interface is even more expansive than previously realized, on account that great quantities of extravesicular material shed from the placenta into the maternal blood. The long-term goal of this application is to elucidate the functions of placental extracellular vesicles in pregnancy. Intercellular communication mediated by extracellular vesicles represents an evolutionarily conserved fundamental mechanism of communication, yet one we know little about. Recent research has informed us on the some of the probable functions of extracellular vesicles in pregnancy. In vitro studies suggest cellular targets of placental vesicles include maternal endothelial and immune cells, and that functions include vascular and immunological adaptation. However, major gaps remain in our understanding of extracellular vesicles, in part because there is a paucity of models available to study placental extracellular vesicles. In this proposal, we close some of these gaps by use of a new in vivo model in we can accomplish the major goals of this application: to distinguish and quantify maternal and placental extracellular vesicles; to identify in vivo maternal cellular targets of placental vesicles; and to observe vesicle-mediated transfer of functional RNA from the placenta to maternal cells. These studies will transform our understanding of placental extracellular vesicles, providing the first evidence of what has so far been phenomenological. The results will inform our development of use of placental vesicles as diagnostic and even therapeutic tools in pregnancy.